Studies on plasmid replication have contributed significantly to our understanding of the regulation of DNA replication. Since bacterial plasmids are small DNA molecules tha replicate autonomously, they serve as useful systems for studies on the mechanism as well as regulation of DNA replication. Plasmids are intimately associated with antibiotic resistance and toxin production in a number of pathogens. Hence studies on plasmids are of considerable medical significance as well. We are investigating the mechanism and regulation of replication of a small Staphylococcus aureus plasmid, pT181. This plasmid is 4.4 kb in size and it encodes tetracycline resistance. We have isolated the pT181-encoded RepC protein that is required for its replication. We have also shown that RepC has a DNA relaxation activity that is specific for the pT181 origin. In addition, Repc creates a site-specific nick within the origin. The role of RepC protein in initiation, elongation and termination will be investigated by using anti-RepC antibodies to inhibit in vitro replication of pT181. Thermo-labile RepC protein will be isolated from strains carrying the plasmid pSA0301 and used for the determination of its role in termination, if any. By site-specific mutagenesis of the origin, nucleotides that are critical in replication will be identified. In vitro deletion analysis of the origin will be carried out to identify the minimal region that is required for replication and relaxation/nicking by RepC protein. In vitro replication experiments will be carried out in the presence of dideoxy NTPs, and the start-site of replication will be identified by 3' to 5' deletion analysis of the partially-replicated origin region using T4 DNA polymerase (in the absence of dNTPs). Mutants of RepC will be isolated by site-directed mutagenesis and studied for their DNA replication, relaxation/nicking and DNA binding properties. Amino acids in RepC that are critically associated with these activities will be identified. A radioimmunoassay using anti-RepC antibodies will be carried out to determine the amount of RepC in a number of pT181 copy mutants and to detect any direct relationship between the amount of RepC and the copy number.